Glioblastoma multiforme (GBM) is the most common and most aggressive type of brain tumor with a median survival of 14 months. Currently, the diagnosis of GBM requires surgical resection and pathological examination followed by concurrent chemoradiotherapy then adjuvant chemotherapy using oral temozolomide (TMZ). Despite this treatment, most tumors recur within the irradiation field, thus GBM continues to represent a significant therapeutic challenge. As such we have focused on the development of drugs that can sensitize GBM to irradiation. We and others have shown that multiple HDACi from diverse chemical backgrounds can sensitize tumor cells to radiation (RT). Our previous work focused on valproic acid (VPA) for several reasons: it is currently used in the clinic as an anti-convulsant; VPA pharmacology including its blood-brain-barrier (BBB) penetrance is well known; and VPA has a low cost per dose. Our group was the first to show that VPA had HDACi activity that could sensitize glioma cells to irradiation both in vitro and in vivo and that maximal radiosensitivity occurred when the HDACi was present both prior to and following irradiation. This preclinical data formed the foundation for our recently published clinical trial of VPA/RT/TMZ in patients with newly diagnosed GBM. The logical next step would be to initiate a randomized trial of VPA/RT/TMZ through one of the cooperative groups. However, that trial is currently not planned. It is not planned for several reasons, including a lack of understanding of how the tumor molecular classification (i.e. MGMT(+) versus MGMT(-)) from the patients in our study affected VPA-induced radioresponse. Second, newer HDAC inhibitors may have improved pharmacokinetics and thus be more effective with less toxicity than VPA. Third, there is a question whether VPA and TMZ have a synergistic toxic effect against megakaryocytes leading to thrombocytopenia. My group is addressing these pre-clinical issues in Project 1 Aim 1. There was also a concern that the addition of a radiation sensitizer to the RT/TMZ combination would lead to late CNS effects in long term survivors, which will be addressed in Project 2. In addition to HDAC inhibitors, there are other classes of molecules that can sensitize GBM to irradiation that will be studied in our preclinical models including two that should progress to clinical trial shortly. The first is the DNA-pK inhibitor from Vertex Pharmaceuticals shown to enhance GBM radiosensitivity both in vitro and in vivo. The second is the XPO1 inhibitor Selinexor. Additionally, we are interested in drugs that are known to cross the blood-brain-barrier but are not currently known as radiation sensitizers. We will screen these drugs for an enhancement of radiosensitivity through an off-target effect through a collaboration with NCATS. As most GBM fail within the first year, there is a need for the development of models of recurrent GBM to test novel therapies. As re-irradiation (re-RT) is often offered as a therapeutic option, additional studies are needed to understand the effects of re-RT in pre-clinical models. The Tofilon lab has recently developed a model of primary GBM RT failure that we will expand to study re-irradiation of recurrent GBM. Questions of dose, fractionation and the addition of radiation sensitizers will be addressed in these pre-clinical models and used to guide future clinical trials. As brain tumors are difficult to evaluate through serial biopsy, the development of surrogate biomarkers from biofluids or imaging would potentially offer information about the tumor that is currently unavailable. Since the last site visit, our evaluation of novel GBM biomarkers has been focused on molecular imaging. As it is assumed that gliomas bypass standard glycolysis through the Warburg effect and that the metabolism of different gliomas contributes to the efficacy of our current therapy, our biomarker studies will focus on the use of 13C hyperpolarized molecular imaging in both the primary and recurrent GBM models. Thus, my section is focused on the pre-clinical development of radiation sensitizers for both primary and recurrent GBM, the use of novel metabolic imaging, and the translation of these pre-clinical findings into clinical trials. Specific Research Aims: 1. To continue the development of VPA as a radiation sensitizer. To determine if the molecular characteristics of GBM tumors are predictive of VPA-RT response we will extend our previous pre-clinical studies by exploring the role of VPA as a radiation sensitizer in MGMT(-) GSCs. This will include both in vitro and in vivo studies. As newer HDACi may have improved pharmacology compared to VPA, new HDAC inhibitors (128) will be screened in combination with RT in a collaboration with the National Center for Advanced Translational Sciences (NCATS) program using a post-IR ?-H2AX screen. Furthermore, additional toxicology including work on megakaryocyte dysfunction will include both TMZ and VPA/TMZ combinations. 2. To test novel radiation sensitizers in models of primary GBM. In order to maximize the potential for Selinexor to be successfully translated to the clinic we will perform additional preclinical work including combination studies with TMZ, studies of the timing of Selinexor with irradiation and tumor pharmacokinetic studies using RNAscope. The DNA-pK inhibitor VX-984 will also be studied in our pre-clinical orthotopic models using a clinically relevant combination of RT/TMZ. An additional screen of all FDA approved BBB penetrant drugs will be conducted in collaboration with NCATS to identify agents already known to penetrate the BBB that can act as radiosensitizers. 3. The development of a model for GBM re-irradiation. Questions from our clinical experience that will be tested in the GBM re-RT model include: does more dose of re-RT improve overall survival; is a single or fractionated dose more effective; does the timing of re-RT after failure affect survival (early versus late); and lastly does the addition of TMZ or novel radiation sensitizers in the setting of reirradiation improve overall survival?